Craniofacial abnormalities are characteristic of embryonic exposure to alcohol. In typical fetal alcohol syndrome poor development of a number of facial features, all of which are derived from the cranial neural crest, is observed. This grant seeks to establish a zebrafish model to study the craniofacial defects associated with embryonic alcohol exposure, and to establish that such environmental insults produce craniofacial defects by interfering with normal signals that control growth. We will first establish what the optimal dose and time schedule for alcohol application is, with respect to deficits in cranial neural crest cells, taking into consideration doses which would be relevant to fetal alcohol syndrome. The zebrafish is an excellent model for comparing teratogens, like alcohol, with genetic defects, to determine what candidate genes might be altered by environmental conditions. In addition, it is possible to overexpress genes of interest by directed injection or transgenesis. The defects in fetal alcohol syndrome partially overlap with some features of holoprosencephaly, which is typically a more dramatic malformation of the central nervous system with associated midline facial features, arising from both genetic and environmental factors. One such genetic cause is a heterozygous mutation in the human Sonic Hedgehog gene. Partial inhibition of Sonic hedgehog in the chick embryo using function-blocking antibodies results in a phenotype similar to a mild holoprosencephaly that is intriguingly similar to fetal alcohol syndrome. The morphological similarity between embryos exposed to alcohol and partial inhibition of Sonic hedgehog suggests a potential mechanistic link. Zebrafish have multiple hedgehog genes, so it is possible that alcohol interferes with a common signaling pathway. To test the hypothesis that ethanol leads to cranial neural crest cell death via a decrease in the availability of zebrafish hedgehog genes, the experiments described in this grant will examine the effect of ethanol on the message levels of genes in the hedgehog signaling pathway. We will also examine the fate of cranial neural crest cells, as well as the growth of the craniofacial structures. We will further attempt to rescue the cranial neural crest cells after ethanol treatment by application of exogenous Sonic hedgehog. These experiments will lay the groundwork to expand our understanding of the mechanisms of craniofacial defects following embryonic exposure to alcohol.